Protocols for Afibril formation in-vitro
Method 1 - “On-line”
Thioflavin T (ThT) is present in the reaction mix during fibrillation process,

monitored continuously by fluorometric measurement.
Reaction is preformed in untreated, white 96F-well plates (Nunc Roskilde,

Denmark, cat # 236108) covered with sealing tape (Nunc, cat # 232701).
1. A stock - A purchased from Biosource, Camarillo, CA, USA (cat # 03-138) or
Sigma, Jerusalem, Israel (cat # A1075), dissolved in DMSO to the final
concentration of 1.8 mM, sonicated (three times 3sec pulses), filtered through 0.2
m filters (PALL, Life Sciences, NANOSEP MF 0.2 m, cat # ODM02C34) and
stored in aliquots at –70oC.
2.ThT stock – ThT purchased from Sigma, Jerusalem, Israel (cat # T3516-5G),
dissolved in 50 mM glycine–NaOH buffer, pH 8.5, to the concentration of 5 mM.
Stored at room temperature covered with aluminum foil. At the day of experiment
prepare working solution of 1.25µM in 50 mM glycine–NaOH buffer, pH 8.5.
3. PBS-Azid- 10mM Na-Phosphate Buffer, pH 7.4, Na-Azid 0.02 %.
Assay:
1. In the well of multiwell plate add in the following order:
A (to the final concentration of 200µM in 20 l).
-
PBS-A up to 20µl.
-
2. Preincubation at room temperature for 20 minutes.
3. Add 80 µl ThT (to the final concentration of 1µM.in the total assay volume of
100µl)
4. Insert plate to a spectrofluorometer (Tecan, Maennedorf, Switzerland).
* Setting the spectrofluorometer for reading:
Measurement parameters: excitation, 450 nm; emission, 485 nm. Gain 56.
-
Kinetics: 60 cycles with 10 minutes intervals (run time 10 hours).
-
Temperature - 30ºC.
-
Orbital shaking, for 5 min between cycles, normal intensity (~200 rpm).
-
Method 2 –Three steps assay: Stirred A incubation, sampling, ThT
fluorescence assay.
Fibrillation preformed in eppendorf test tubes.

Kinetics of A fibril formation is monitored by sampling the shaked reaction

solution at different time points along the fibrillation process.
Extend of fibrillation evaluated by ThT assay preformed as in Method 1.

Assay:
1. Prepare ThT working solution (20µM in Glycine Buffer 50mM pH 8.5), cover with
aluminum foil.
2. In eppendorf tubes dilute A
42µM in PBS-A from the DMSO stock (Method
1).
3. Incubate at room temperature in the fibril-formation buffer, with shaking (~200
rpm) for several hours.
4. Sampling procedure:
At the indicated times stop shaking. )a
Vortex eppendorf . )b
Take 95 µl of fibril-formation mix to the multiwell plate. )c
Add 5 µl ThT working solution. )d
Mix gently. )e
Read in spectrofluorometer. )f
* Setting the spectrofluorometer for reading:
- Measurement parameters: excitation, 450 nm; emission, 485 nm. Gain 56.
- Read at room temperature.